Several methods exist for system-wide measurement of protein concentrations. In one approach, antibody arrays are used to specifically bind to multiple proteins in complex mixtures. However, optimal binding between protein antigens and antibodies tend to vary widely, making the approach generally unsuitable for proteome-wide analysis. A second method involves the use of heavy isotope standards. The standards are mixed with the protein samples to provide an internal reference for the measurement of protein concentration. These methodologies measure steady-state protein levels and cannot analyze the dynamics of in vivo proteome turnover.
Protein molecules are in dynamic equilibrium in vivo: they are continuously synthesized and degraded during the lifetime of the organism (1, 2). The turnover rate of proteins can vary from minutes to years, often conforming to their biological functions (3, 4). The constant renewal of the protein population is an energy-intensive process, yet it allows the cell to rapidly modulate protein levels in response to changes in the environment (5, 6). Proper proteome dynamics are critical to normal development and maintenance of health (7, 8). For example, the dysregulation of protein turnover has been implicated in the aging process (9), increased degradation of the CTFR chloride channel is a primary cause of cystic fibrosis (10), and the inability to clear protein aggregates leads to pathogenic accumulations in Alzheimer's, Parkinson's, Creutzfeldt-Jakob, and other age-related diseases (11).
The turnover rate of a protein is established by its relative rates of synthesis and catabolism. Thus, the lifetime of a protein is influenced by a number of regulated processes at the level of the cell (transcription, translation, proteolysis, autophagy) and tissue (development and regeneration) as well as its biochemical properties (structural stability, hydrophobicity, sequence motifs) (1, 12-15). The ability to measure turnover rates on a proteome-wide scale can help elucidate the interplay between these factors and identify novel processes that play a role in proteome homeostasis. It can also identify proteins whose dysregulation influences or results from pathological processes.
Traditionally, protein turnover has been studied by measuring the incorporation of radioactive, tracer amino acids into proteins or bulk tissues (16-20). The advent of modern proteomics has enabled scientists to utilize mass spectrometry to detect the incorporation of stable isotopes into proteins (21, 22).